The present invention relates to P. gingivalis polynucleotides and to the use of these polynucleotides in the production of nucleotide probes for detection of P. gingivalis. The polynucleotides may also be used in the production of recombinant P. gingivalis polypeptides. The polynucleotides and recombinant polypeptides can also be used in compositions for use in raising an immune response in a subject against P. gingivalis and treating or preventing or reducing the severity of the condition known as periodontitis.
Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth""s supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is P. gingivalis as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas P. gingivalis is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of P. gingivalis in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of P. gingivalis. These findings in both animals and humans suggest a major role for P. gingivalis in the development of adult periodontitis.
P. gingivalis is a black-pigmented, anaerobic, asaccharolytic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganism has an absolute growth requirement for iron, preferentially in the form of haeme or its Fe(III) oxidation product haemin and when grown under conditions of excess haemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of P. gingivalis including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes.
The present inventors have isolated P. gingivalis polynucleotides sequences which can be used to develop nucleotide probes specific for P. gingivalis. These probes are of particular use in the detection of P. gingivalis infection. Further the polynucleotides can be used for recombinant production of P. gingivalis polypeptides.
Accordingly, in a first aspect the present invention consists in an isolated polynucleotide of at least 20 nucleotides, the polynucleotide comprising a contiguous sequence of at least 20 nucleotides which is identical to a contiguous sequence of at least 20 nucleotides within a sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 1120 and sequences complementary thereto.
In a preferred embodiment of the present invention the polynucleotide is at least 30 nucleotides, preferably at least 50 nucleotides and more preferably at least 100 nucleotides.
In a further preferred embodiment the polynucleotide comprises a contiguous sequence of at least 30 nucleotides which is identical to a contiguous sequence of at least 30 nucleotides within a sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 1120 and sequences complementary thereto, preferably the polynucleotide comprises a contiguous sequence of at least 50 nucleotides which is identical to a contiguous sequence of at least 50 nucleotides within a sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 1120 and sequences complementary thereto, and more preferably the polynucleotide comprises a contiguous sequence of at least 100 nucleotides which is identical to a contiguous sequence of at least 100 nucleotides within a sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 1120 and sequences complementary thereto.
In a second aspect the present invention consists in an isolated polynucleotide, the polynucleotide comprising at least 20 nucleotides, the polynucleotide having a sequence which hybridises under stringent conditions to a sequence selected from the group consisting of SEQ. ID. NO. 1 to SEQ. ID. NO. 1120 and sequences complementary thereto.
It is preferred that the polynucleotide is at least 30 nucleotides, preferably at least 50 nucleotides, and more preferably at least 100 nucleotides.
It is also preferred that the polynucleotide is DNA.
In a preferred embodiment of the present invention the polynucleotide further comprises a detectable label. Non-limiting examples of labels which may be used include biotin, radio isotopes, fluorescent labels etc.
The present invention also provides method for detecting the presence of P. gingivalis nucleic acid in a sample comprising:
(a) contacting a sample with the polynucleotide of the present invention under conditions in which a hybrid can form between the polynucleotide and a P. gingivalis nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a P. gingivalis nucleic acid in the sample.
As will be readily appreciated by those skilled in the art the polynucleotides of the present invention can be used to produce P. gingivalis polypeptides. This may achieved using any of a number of methods well known in the art, however, this will typically involve the use of a recombinant expression vector comprising the DNA of the present invention operably linked to a transcription regulatory element. It is preferred that the DNA is operably linked to a transcription regulatory element. This recombinant expression can be used in the transformation of an appropriate cell.
The transformed cell can then be used to produce P. gingivalis polypeptides. This involves culturing the cell under conditions that permit expression of the polypeptide and recovering the polypeptide.
The nucleotides of the present invention can also be used to raise an immune response in an animal by DNA vaccination. Accordingly the present invention also provides a composition for use in raising an immune response directed against P. gingivalis in a subject, the composition comprising an effective amount of at least one DNA molecule of the present invention and a pharmaceutically acceptable carrier. It is preferred that the pharmaceutically acceptable carrier is an adjuvant.